1. Field of the Invention
The invention concerns a process of in vivo liberation a linear DNA fragment from a vector in order to integrate this fragment into the cellular genome. The invention further concerns the use of this process and the results of this use.
2. Brief Description of the Prior Art
The first breakthrough of reverse genetics 20 years ago was Transgenesis. The transgenesis is the technique which allows to introduce a exogenous DNA sequence into a host cell. For example, DNA micro-injection in an egg fertilized by mammal leads, in a certain number of cases, to the integration of micro-injected DNA molecule into the genome of the fertilized egg. Transgenesis implies that a foreign DNA fragment is introduced into the genome of a multicellular organism and transmitted to progeny. Therefore, the foreign DNA must be present in a stable form in the embryo at an early stage of development in order to be transmitted into progeny.
DNA transfection in mammal cells by means of precipitated calcium phosphate precipitation can also lead, in a certain number of cases, to the integration of the exogneous DNA to the genome of the cell host which is called stable transfection. The exogenous DNA can be introduced into cells under two forms, either linear or circular. When the DNA is introduced under linear form, the linear fragment is prepared in vitro before its introduction into the host cell, generally by excision of the desired fragment with restriction enzyme from a plasmid for example. As regards the DNA under circular form, the introduced DNA is generally a supercoiled plasmid.
All cells have systems of maintenance and repair of their DNA. One of the particular signals of stress which activates the intervention of these systems is the generation of DNA free ends in the cell. The cell has then two solutions to resolve this type of problem:
The first solution is the degradation of the DNA presenting the free ends (for example the case where the cell should eliminate a DNA being able to have a viral activity). This solution is based on the presence in these systems of maintenance and repair of exonucleases, which degrade the DNA by digesting it from the free ends.
An other solution is the recombination of the free ends with the cellular genome. This recombination can take different forms, notably the integration of the exogenous molecule into the genome of the cell.
As a consequence, the works aiming at integrating of the exogenous DNA by introduction of naked double-stranded DNA under circular or linear form meet three types of major problems as well as certain number of collateral problems.
The first one of these problems is the efficiency of the integration of the exogenous molecule in the DNA of the cell host. This lack of efficiency implies the injection of very hight amount of exogenous DNA for a very few number of integration. In some species such as fish, plants or insects, the exonucleases are so efficient that the exogenous DNA is never integrated in the chromosome and it stays episomal. For example, if the circular form is used for the exogenous DNA, a nick is necessary for having integration. Indeed, the integration process needs the presence of a free end. If the linear form is used, most of the exogenous DNA is degraded by the exonucleases due to the presence of the free ends.
The second problem is the integrity of the integrated DNA. The linear fragment of exogenous DNA exposes its free ends to the cellular exonucleases before its arrival to the nucleus. This prolonged exhibition of its ends in the cell limits in a significant way the chances of the exogenous DNA to be wholly integrated into the cellular genome. A way of increasing the chances to be wholly integrated into the genome consists in adding some cohesive single-stranded DNA overhangs at each ends of the exogenous DNA, for example made by digestion with the same restriction enzyme. These cohesive extremities allow the DNA fragments to associate in a multimer and to prevent the complete degradation of the DNA before its integration. Another way of increasing the chances is to surround the DNA fragment to integrate with long and neutral DNA sequences. In the case of the exogenous DNA is included in a supercoiled plasmid, the intregrated fragment comprises not only the exogenous DNA but also the whole vector.
The third problem is the control of the number of integrated copies. The multimerization, deliberated or not, of the exogenous DNA presents the inconvenience to favor the insertion of several copies of the exogenous DNA fragment. Similarly, when the exogenous DNA has a circular form, the plasmid is integrated as a concatemer. This multiple insertion has for consequence to introduce a chromosome instability and to result in problems of regulation of the expression due to the presence of the same gene in multiple copies.
Transgenesis is, more than ever, an essential tool for biologists. The study of human diseases relies to a great extent on the use of animal models. Vast gene sequence informations are available for the pharmaceutical industry. These informations would provide new targets for drug. However, the function of the majority of the genes and related proteins in an organism remains unknown and the efficiency of target validation does not appear to have significantly changed.
Genetic studies and structural genomics have shown that biochemical pathways and physiology are highly conserved throughout the animal world. Animal models can therefore be used to investigate processes relevant to human diseases. Animal model can be used to efficiently identify and validate optimal screening targets. Screening target selection can be improved and will lead to fewer failures and a more efficient drug development process.
Mouse is a well known mammalian model which is abundantly used. The most widely used method for the production of transgenic animals is the microinjection of DNA into the pronuclei of fertilized embryos. This method is rather efficient for the production of transgenic mice but is much less efficient for the production of large transgenic mammals such as cows and sheep. Moreover, the transgenic animals from the available transgenesis method are often mosaic for the transgene resulting in the lack of transmission of the transgene to the progeny. Some animals present a high resistance to transgenesis such as fish or bird. Among them, the problem of fish transgenesis is more detailed below.
Tank fish have risen to high popularity as vertebrate models in developmental biology and genetics. Indeed, the zebrafish (Danio rerio) is a popular model system for vertebrate developmental studies because it offers the opportunity to combine classical genetic analysis with an easily accessible and manupilable embryo. Genetic studies of the zebrafish benefit from the 2–3 month generation time, the ability of females to routinely lay hundreds of egg, and the small size of the adults. Embryological studies benefit from the large, transparent embryos.
However, the usefulness of tank fish is still limited by the lack of some methodological tools, above all a simple and efficient technology for transgenesis, which has become a major technique in fundamental research and has varied applications in agronomy and biomedecine. In particular, attempts to establish embryonic stem (ES) cells in fish as cellular vectors for transgenesis, have so far been unsuccessful.
Therefore, the method of choice to generate transgenic fish remains the injection of high concentrations of DNA (approximately 106 plasmid copies) in the cytoplasm of one cell-stage embryos. Plasmids have been microinjected in linearized and circular form, and in both types of experiments transgenesis has been achieved. This technology is fast and easy, due to the transparency and great size of most fish eggs, but unfortunately, it is also rather inefficient, with a frequency of genomic integration and germline transmission which usually lies in a range of a few percents [Stuart et al., 1988 Development 103, 403–412; Stuart et al., 1990, Development 109, 577–584; Culp et al., 1991, Proc Natl Acad Sci USA, 88, 7953–57; Lin et al., 1994 Dvelopmental biology 161, 77–83; Collas et al., 1998 Transgenic Research, 7, 303–309]. Recent studies have shown that this is likely to be due to late and mosaic integrations: DNA persists in an unintegrated form in the egg cytoplasm and is inherited only by a subset of blastomeres. After injection, the plasmid sequences are transiently amplified and form long concatemers consisting of many unit-length copies of the plasmid arranged in tandem (Stuart et al. 1988, supra). The foreign DNA usually inserts into one site in the host genome but usually consists of tandem arrays of the original injected construct (Culp et al., 1991, Supra).
It is generally accepted that increasing the frequency of transgenic fish generated by plasmid microinjection is difficult. Injecting higher amounts of DNA is toxic to the embryos so one must attempt to improve the efficiency of integration. Several attempts to improve the rate of integration and transmission of transgenes have been performed. For example, the use of DNA-NLS complexes has been reported, although most authors found no improvements with this technology [Liang et al., 2000 Mol Reprod Dev 55, 8–13]. In principle, technologies using flanking repeats of adeno-associated virus [Fu, 1998 Nature Biotech, 16, 253–257] or of transposons [Izsvak et al., 2000 J Mol Biol 302, 93–102] may also increase transgene integration. However, to the best knowledge of the inventors, positive results have not been not reported with these techniques, which also suffer from the potentially deleterious presence of repeats in the plasmids. Moreover, these vectors are limited for the size of the DNA sequences that can be engineered into them.
Therefore, numbers of methods have emerged to improve and develop new ways to increase the transgenesis efficiency. Nevertheless, a very limited amount of methods allows better control and numbers of species remain resistant or very inefficient for this technology. Main problems encountered are:                Transgenesis control and efficiency        Early integration events (germ line transmission)        The control of integrated copy number.        